AAV is a small (25 nm) single-strand DNA virus that is ubiquitous in nature. Initial characterizations of this virus demonstrated that it is non-autonomous since replication and production relies on helper functions provided by other viruses including adenovirus (hence its name) [1]. AAV is a simple virus composed only of a non-enveloped protein capsid and a single-strand DNA molecule that is flanked at each end by T-shaped ITRs. The AAV ITRs provide necessary functions for AAV production and transduction including acting as the viral origin, capsid packaging, second-strand synthesis in the host cell, and genome persistence [1]. Regarding the viral capsid, many serotypes have been identified that alter cell tropism due to the use of different cellular receptors [2].
The first AAV vector was generated over 30 years ago by the demonstration that the ITRs of AAV serotype 2 (ITR2) confer the abilities of plasmid replication and single-strand DNA capsid packaging in the presence of the AAV replication and capsid genes, along with the required adenovirus helper functions [3, 4]. This seminal observation resulted in the ability to produce AAV transgenic DNA vectors in which the only viral DNA sequence are the ITRs, with a remaining 4.5 kb sequence of choice [1]. Since that initial report, AAV vectors are now among the most promising methods of gene delivery for the treatment of human genetic diseases. To date, over 80 clinical trials using AAV vectorology have been performed and all have utilized the ITR2 sequence necessary for transgenic DNA replication, packaging, second-strand synthesis and persistence [1].
Due to the technical difficulties of ITR synthesis, only a few reports in the last 32 years have demonstrated the ability to synthesize functional mutant ITR2 sequences capable of AAV vector production and transduction [5-8]. In all of these cases, the generated ITR sequences were based on the natural ITR isolate from AAV serotype 2 and, in a single recent report, hybrid ITRs using the natural ITR sequence of AAV serotype 5 [5]. An additional study compared the wild type ITRs of AAV serotypes 1-6 at the levels of production and in vivo transduction [10]. The collective conclusion of that work was that the natural ITRs (1-6) did not significantly affect vector production or the transduction efficiency in mouse liver [10].
The present invention provides alternatives to ITR2 that have improved characteristics and are suitable for generating vectors with a wide variety of uses, including gene therapy.